KR20130030309A - Cell culture method and screening method - Google Patents

Abstract

It is an object of the present invention to provide a cell culture method capable of maintaining a function in vivo for a long time and culturing in a minimum required cell number. In the cell culture method according to the present invention, the differentiated cells are obtained by culturing in a state where the undifferentiated cells are stratified in the divided microspace. For screening pharmaceuticals, undifferentiated cells that differentiate into liver cells, intestinal epithelial cells, nerve cells, cardiomyocytes, and vascular endothelial cells are preferred. In particular, when making predictions on humans such as drug dynamics, human cells are preferable.

Description

Cell culture method and screening method {CELL CULTURE METHOD AND SCREENING METHOD}

The present invention relates to a cell culture method and a screening method.

The use of cells isolated from tissues for testing and testing has become an indispensable method in the field of biotechnology. It is widely used for diagnosing diseases and conditions, searching for new drugs and determining drug efficacy, or for testing animals, testing plants, and testing environmental pollutants. For this reason, cell types used in the field of biotechnology are diversified.

Isolated cells are often used for testing in a suspended state, but in most cases, they are cultured in a state of being adhered to a culture dish and used for various tests and examinations. The primary cells and chemotaxis cells used for cell culture are required to exhibit the same drug sensitivity, toxic response, and the like as the so-called in vivo tests. In other words, similar cell functions in vivo are required on cell culture vessels. Moreover, since isolation operations for obtaining primary cells are complicated and cell culture strains used for cell culture tests are expensive, a test method with a small cell number is desired.

Recently, in drug development, the suspension of development in the clinical trial stage has become a problem. This is due to animal species differences in the pharmacokinetic test phase. Until now, in the preclinical pharmacokinetic test, animals such as rats, dogs, and monkeys have been used to predict the pharmacokinetics of the drug. However, in clinical trials using humans, it is clear that the prediction is virtually inconclusive. Therefore, the prediction of humans such as pharmacokinetics is the most effective and convenient way to use human samples, and is important for efficient drug development and safe clinical trials.

In the pharmacokinetic test for examining the pharmacokinetics of a drug, it is mainly absorption, metabolism and excretion in the liver, and human samples used are liver slices, liver cells, liver microsomes and the like. Among these, soy sauce slices are difficult to obtain, and liver microsomes can only be subjected to metabolic tests using only limited metabolic enzymes. Therefore, the use of liver cells is considered to be the most effective.

In the screening, the culture dish used is each plate of resin chalena, 6 wells, 12 wells, 48 wells, 96 wells. These generally have almost the same size of the entire plate, so that the larger the number of wells, the smaller the size of one well. This one well corresponds to one culture dish. In addition, in the recent flow of micronization furnaces, 384 well plates, which are more small-diameter and composed of a large number of culture dishes, have also started to be used, and those adapted to the desired screening method are used. The bottom part of these culture dishes is a flat plate shape, and this bottom surface is used as a culture surface.

However, when conventional culture vessels are used for culturing tissue cells, the original function may be lost and dedifferentiated, or undifferentiated cells may not be differentiated, and the problem of not expressing the target cell function is a problem. . For example, when human fresh liver cells are cultured on a conventional flat plate, the metabolic enzyme function when isolated is significantly lowered in about 1 day. Therefore, the drug metabolism test is performed within 4 hours after cell seeding on the plate. There is a case. That is, there are problems that cannot be used for testing while prolonged incubation, and can not examine the long-term metabolic stability.

In order to solve the above problems, attempts are made to coat human or animal-derived biological materials (sugar proteins, proteins, etc.) on the culture vessel surface (see Patent Document 1), or attempts to culture in a polymer gel (see Patent Document 2); Attempts have been made to form liver cell masses in micro containers (see Patent Document 3).

Japanese Patent Application Laid-open No. Hei 8-319317 Japanese Patent Laid-Open No. 8-308562 International Publication No. 2008/130025

However, in the method disclosed in Patent Literature 1, there are problems such as high cost, difficulty in forming uniform cell aggregates in a culture vessel, and inability to maintain in vivo functions for a long time because the biological material to be coated is special. have. Also in the method disclosed in Patent Document 2, there are problems such as being unable to control the size of the cell aggregate, being unable to easily observe the microscope, and having complicated operability as a screening substrate. In addition, since both methods mentioned above use commercially available dishes and plates as support containers, efficient screening at the required minimum cell number is difficult. Even in the method disclosed in Patent Literature 3, it is possible to improve liver cell metabolic activity in the early stage of culture, but it is difficult to maintain metabolic activity for two weeks or more.

It is an object of the present invention to provide a cell culture method capable of maintaining a function in vivo for a long time and culturing in a required minimum cell number.

In the cell culture method according to the present invention, the differentiated cells are obtained by culturing the undifferentiated cells in a multilayered state in a partitioned microspace. Here, stratification means that the lamination of cells occurs in two or more layers. In the case of screening pharmaceuticals, undifferentiated cells which are differentiated into hepatocytes, intestinal epithelial cells, nerve cells, cardiomyocytes and vascular endothelial cells are preferred. In particular, when making predictions about humans such as drug dynamics, the undifferentiated cells are preferably human cells. The undifferentiated cells are preferably liver cells, progenitor cells and the like.

Moreover, it is especially preferable that the said divided micro space is the said micro container in the cell culture container which has a some micro container on the surface. Here, it is preferable that the bottom area of the said microcontainer is 9x10 <^-4> mm <^2> -9x10 <^-2> mm <^2> , the height of a side wall is 15 micrometers-300 micrometers, and the width | variety of a side wall is 3 micrometers-15 micrometers. Moreover, in order to make microscope observation easy, it is preferable that the area | region in which the micro container in the said cell culture container was formed has transparency.

The screening method according to the present invention is to screen a compound by arranging a plurality of cells cultured by the above culture method. Moreover, in order to culture | cultivate with required minimum cell number, it is preferable that the said cell culture container has two or more partitioned spots which consist of a plurality of said micro containers.

According to the present invention, it is possible to provide a cell culture method capable of maintaining the function in vivo uniformly for a long time and culturing in the required minimum cell number.

BRIEF DESCRIPTION OF THE DRAWINGS It is a top view which shows the structure of the cell culture container used for the cell culture method example which concerns on Example 1. FIG.
FIG. 2 is a cross-sectional view showing the structure of a cell culture vessel used in the cell culture method according to Example 1. FIG.
It is a top view which shows the structure of the cell culture container used for the screening method which concerns on embodiment.
It is sectional drawing which shows the structure of the cell culture container used for the screening method which concerns on embodiment.

In the present invention, undifferentiated cells, such as liver stem cells, are cultured in a state of being multilayered in a uniform size in a microcontainer that is a divided microspace. Of particular importance in the present invention is the use of undifferentiated cells with proliferative capacity, culturing in compartmentalized microspace, and culturing in multiple layers. Thereby, for example, a cell mass containing differentiated hepatic parenchymal cells can be formed, and the function can be improved. In addition, since liver stem cells proliferate undifferentiated, the function of the cells can be maintained. The present invention is such a culture method and a screening method using the same.

On the surface of the culture vessel used in the culture method and the screening method according to the present invention, an uneven pattern, that is, a plurality of micro containers, that is, a culture space is formed. By optimizing the width and height of the side walls (convex portions) for partitioning the micro containers, cells can be cultured only in the micro containers to maintain a uniform differentiation state. It is also conceivable to form a partitioned culture space made of gel instead of a micro container.

The dimensions of the microcontainers surrounded by the side walls need to be in the optimum range for culturing the cells. If the bottom surface area of the microcontainer is too large, the cells elongate partially in the same manner as the culture on the plate, and do not form a uniform layered state. On the other hand, if the bottom area of the microcontainer is too small, the cells cannot be accommodated. Therefore, the size of the space is preferably in a range that can accommodate a plurality of dozens depending on the cell species to be cultured.

In addition, the sidewall of the microchamber needs to be in an optimum range for culturing cells. If the sidewall is too wide, the cells adhere to the top surface of the sidewall and are not suitable for culture. If the width of the side wall is too narrow, fabrication becomes difficult. If the height of the side wall is too low, the cells will cross the side wall and are not suitable for culture. If the height of the side wall is too high, not only production is difficult, but also material diffusion becomes difficult and the culture environment is deteriorated.

EMBODIMENT OF THE INVENTION Below, embodiment of this invention is described. However, the present invention is not limited to the following embodiments. In addition, the following description and drawings are suitably simplified for clarity of explanation.

Embodiment

The structure of the micro container of the cell culture part used for the cell culture which concerns on embodiment is demonstrated using FIG. FIG. 1 is a plan view showing the structure of a microcontainer according to the present embodiment, and FIG. 2 is a II-II cross-sectional view of FIG. 1. As shown in FIG. 1, the cell culture part 10 is equipped with the micro container 11 and the side wall 12. As shown in FIG. In the culture surface of the cell culture unit 10, a plurality of side walls 12 are formed in a mesh shape, and a space surrounded by all sides by the side walls 12 serves as the micro container 11.

b

15 ㎛ , 또한, c/b

2 로 할 필요가 있다. 측벽 (12) 의 폭 (b) 이 15 ㎛ 를 초과하면, 측벽의 상면에 세포가 접착되어, 배양에 적합하지 않다. 한편, 측벽 (12) 의 폭 (b) 이 3 ㎛ 미만에서는, 제작이 곤란해진다. 측벽의 높이는 너무 낮으면, 세포가 측벽을 넘어가 버려, 배양에 적합하지 않다. 측벽 (12) 의 높이 (c) 가 측벽 (12) 의 폭 (b) 의 2 배 미만이면, 마이크로 용기 (11)에서 배양하는 세포가 타고 넘어가, 인접하는 마이크로 용기 (11) 로 이동한다. 또, 구체적으로는, 1 변 100 ㎛ 의 사각형 마이크로 용기 내에서 인간 태아 간 세포를 중층화시키는 경우, 측벽 (12) 의 높이 (c) 는 15 ㎛ ～ 300 ㎛ 가 바람직하고, 50 ㎛ ～ 150 ㎛ 가 더욱 바람직하다. 여기에서, 측벽의 높이 (c) 가 너무 높으면, 제작이 곤란할 뿐만 아니라, 물질 확산이 되기 어려워져 배양 환경이 악화되고 만다. 측벽 (12) 은 다단계 형상이어도 된다.In FIG. 1, the width a of the bottom surface of the micro container 11, the width b of the side wall 12 for partitioning the micro container 11, and the height c were shown. Where 3 μm

b

15 μm, also c / b

Must be 2 When the width b of the side wall 12 exceeds 15 micrometers, a cell adheres to the upper surface of a side wall, and is not suitable for culture | cultivation. On the other hand, when the width | variety b of the side wall 12 is less than 3 micrometers, preparation becomes difficult. If the height of the side wall is too low, the cells will cross the side wall and are not suitable for culture. When the height c of the side wall 12 is less than twice the width b of the side wall 12, the cells cultured in the micro container 11 are carried over and move to the adjacent micro container 11. In addition, specifically, when layering human fetal liver cells in a 100 micrometer square micro container, the height c of the side wall 12 has preferable 15 micrometers-300 micrometers, and 50 micrometers-150 micrometers More preferred. Here, when the height c of the side wall is too high, not only production is difficult but also material diffusion becomes difficult and the culture environment is deteriorated. The side wall 12 may have a multistage shape.

The shape of the bottom surface of the micro container 11 is not particularly limited, and various shapes other than square, circle, and polygon may be adopted. As for this bottom surface area, 9x10 <^-4> mm <^2> ~ 9x10 <^-2> mm <^2> is preferable. Moreover, an isotropic shape is preferable and when a bottom surface is rectangular shape, it is preferable that a long side is 1 to 1.5 times of a short side.

In order to minimize the required number of cells, the cell culture part used in the present invention may have partitioned spots composed of as many microcontainers as necessary for one screening, as shown in FIGS. 3 and 4. For example, using a microcontainer having a square of 200 μm on one side with high differentiation efficiency and having a height of 50 μm, if the minimum number of cells required for screening is about 1000, nine microcontainers are needed. By forming the spots to be partitioned and forming a plurality of spots again, high throughput screening that enables the inspection of a plurality of reagents and medicines at the same time becomes possible.

Here, FIG. 3 is a top view which shows the structure of the other cell culture part which concerns on this embodiment, and FIG. 4 is sectional drawing IV-IV of FIG. 3, the side wall 14 which partitions a some micro container, and the partitioned spot 13 are shown. The height d of the side wall 14 should just be a capacity | capacitance which can keep supernatant liquids, such as a culture liquid and a reaction liquid, from drying, and should just set suitably.

Although it does not specifically limit as a method of manufacturing the uneven | corrugated pattern of the cell culture part of this invention, For example, transfer molding using a mold, three-dimensional light shaping | molding, precision machine cutting, wet etching, dry etching, laser processing, electrical discharge machining, etc. The method can be mentioned. It is desirable to appropriately select these methods of preparation in view of the use of the cell culture vessel, the processing precision required, the cost and the like.

As a specific example of the transfer molding method using a mold, a method of forming an uneven pattern by resin molding using a metal structure as a mold is mentioned. This method is preferable because the shape of the metal structure can be reproduced in an uneven pattern in the resin at a high transfer rate, and the material cost can be lowered by using a general-purpose resin material. The method of using the mold of such a metal structure is low cost, and is excellent in the point which can satisfy high dimensional precision.

As a manufacturing method of the said metal structure, the plating process, the precision machine cutting, wet etching, dry etching, laser processing, for the resist pattern produced by photolithography or the resin pattern produced by 3D optical molding, for example, Electrical discharge machining; and the like. What is necessary is just to select suitably in consideration of a use, required processing precision, cost, etc.

As a method of forming an uneven pattern in resin using the metal structure obtained above as a template, roll transfer by injection molding, press molding, monomer cast molding, solvent cast molding, hot embossing molding, extrusion molding, for example. Etc. can be mentioned. It is preferable to employ injection molding from the viewpoint of productivity and mold transferability.

The material constituting the screening chip of the present invention is not particularly limited as long as it has self-supportability, and examples thereof include synthetic resins, silicones, and glass. It is preferable to use transparent synthetic resin as a material from a viewpoint of cost visibility and the cell visibility by microscope observation.

As transparent synthetic resin, acrylic resin, such as polymethyl methacrylate and methyl methacrylate-styrene copolymer, styrene resin, such as polystyrene, olefin resin, such as cycloolefin, polyethylene terephthalate, polylactic acid, for example Ester resins such as these, silicone resins such as polydimethylsiloxane, polycarbonate resins, and the like. Such resin may contain various additives, such as a coloring agent, a diffusing agent, and a thickener, in the range which does not impair transparency.

The screening chip of this invention aims at improving the surface hydrophilicity, biocompatibility, cell affinity, etc., and may surface-treat on the uneven | corrugated pattern surface side, and the modified layer and / or coating layer may be arrange | positioned.

The method for forming the modified layer is not particularly limited unless it is a method of losing self-supportability or causing extreme surface roughness of 100 μm or more. For example, chemical treatment, solvent treatment, or surface graft polymerization may be used. And chemical treatments such as introduction of graft polymers, physical treatments such as corona discharge, ozone treatment, and plasma treatment.

The method of forming the coating layer is not particularly limited, and examples thereof include dry coating such as sputtering and vapor deposition, wet coating such as inorganic material coating, and polymer coating.

On the uneven pattern, it is preferable to impart hydrophilicity in order to inject the culture solution without mixing bubbles, and inorganic vapor deposition is preferable as a method of forming a uniform hydrophilic film.

Moreover, when cell affinity is considered, it is more preferable to coat cell affinity proteins, such as collagen and fibronectin, for example. In order to coat a collagen aqueous solution etc. uniformly, it is preferable to coat, after forming the hydrophilic membrane mentioned above. Usually, in cell culture, since the culture on the surface of the extracellular matrix is desired to mimic the in vivo environment, after the uniform hydrophilic inorganic membrane is arranged as described above, an organic membrane made of a suitable extracellular matrix is placed on the cultured cells. Is particularly preferred.

The cells cultured by the culture method and the screening method of the present invention are preferably undifferentiated cells, hepatic stem cells, hepatic progenitor cells, intestinal stem cells, intestinal epithelial progenitor cells, mesenchymal stem cells, myocardial progenitor cells, embryonic liver cells These may be mentioned, and what is necessary is just to select a cell type suitably according to the objective of screening. For example, for the purpose of screening assuming a metabolic reaction in the liver of a medicine, liver stem cells or the like that are differentiated into liver cells are used. In particular, human liver stem cells are used for the purpose of metabolic reaction of pharmaceuticals in humans.

In the screening method of the present invention, it is necessary to seed the appropriate cell number in order to place the cells only in the microchamber for culturing the cells and to express similar functions in vivo in the space. The cell seeding density is preferably 1.0 × 10 ⁴ to 5.0 × 10 ⁶ cells / cm 2. For example, when a micro container is square and one side is 100 micrometers, 1.0 * 10 <^4> -1.0 * 10 <^5> cells / cm <2> is preferable.

When the cells to be layered are liver cells or intestinal epithelial cells, it is preferable to measure and screen gene expression, metabolic enzyme activity, transporter activity and the like.

In addition, when the cells to be layered are neurons or cardiomyocytes, it is preferable to measure and screen gene expression amounts, enzyme activity action potentials, and the like.

When the cells to be stratified are vascular endothelial cells, it is preferable to visually screen angiogenesis.

Example

Next, although the Example of the cell culture method which concerns on this invention is described, this invention is not limited to these Examples.

<Cultural Culture of Fetal Liver Cells>

Frozen stocks of human fetal liver cells were seeded on type IV collagen coat dishes (manufactured by BD), incubated for 10 days, and expanded. Subsequently, on the uneven pattern substrate coated with 0.03% type IV collagen (manufactured by Nitta Gelatin), liver cells were seeded at a rate (3.8 × 10 ⁴ cells / cm 2) in a single microcontainer and 5% CO ₂ And incubated at 37 ° C. for 3 weeks. The composition of the culture used was 10% fetal bovine serum, 1 μg / ml insulin, 1 × 10 ⁻⁷ M dexamethasone, 10 mM nicotinamide, 2 mM L-glutamine, 50 μm β-mercaptoethanol, 5 in DMEM / F12 medium. mM HEPES, 59 μg / ml penicillin, 100 μg / ml streptomycin was added, and from day 1 after sowing, 25 ng / ml HGF, 20 ng / ml EGF, 10 ng / ml on costin M was added to the culture medium again. It was added and used. Medium exchange was performed every few days using 0.5 ml of fresh medium of the same composition.

Gene expression analysis

Gene expression of cytochrome P450 (CYP), a target metabolic enzyme in the liver, was evaluated by recovering RNA from cells cultured for a predetermined number of days, and performing real-time PCR after cDNA synthesis.

Example 1

As an uneven | corrugated pattern shape shown in FIG. 1, the pattern of a = 100 micrometers, b = 10 micrometers, and c = 50 micrometers was produced by photolithography, Ni electroplating was performed, and the metal mold | die which has the corresponding uneven | corrugated shape was obtained. Pattern transfer was performed on polystyrene by hot embossing using this metal mold, and the resin base material of the said dimension was produced. A 100 nm silicon dioxide film was formed on the surface of the resin substrate by vacuum deposition, gamma ray sterilization was performed to obtain an uneven pattern substrate. After coating IV collagen on the uneven substrate, human fetal liver cells were cultured.

Comparative Example 1

A planar 24-well culture plate with commercially available (Becton Dickinson, Falcon (registered trademark)) completes the γ-ray sterilization was used, and human fetal liver cells were cultured after coating with type IV collagen.

In Table 1, the gene expression amount of CYP3A4, CYP2D6, CYP2C9 after 3 weeks of culture in Example 1 and Comparative Example 1 is shown. In the table, the value which set each CYP expression amount after three weeks of the comparative example 1 was 1. In Example 1, the expression amount was higher than that of Comparative Example 1 in any CYP, and the function was maintained even in culture for 3 weeks.

                                  Example 1 Comparative Example 1
CYP3A4 4.5 1.0

CYP2D6 8.5 1.0

CYP2C9 2.3 1.0

Industrial availability

The present invention can be applied to a cell culture method for culturing cells isolated from tissues.

10 cell culture
11 micro containers
12 sidewalls of micro containers
13 spots
14 spot sidewalls

Claims (6)

A cell culture method in which a differentiated cell is obtained by culturing in a layered state in undifferentiated cells obtained from fetal liver tissue in a partitioned microspace. The method of claim 1,
The cell culture method characterized in that the undifferentiated cells are human cells. The method of claim 1,
Said micro space is the said micro container in the cell culture container which has a some micro container on the surface, The area | region in which the said micro container was formed has transparency, The cell culture method characterized by the above-mentioned. The method of claim 3, wherein
The bottom area of the said microcontainer is 9x10 <^-4> mm <^2> -9x10 <^-2> mm <^2> , the side wall height is 15 micrometers-300 micrometers, and the side wall width is 3 micrometers-15 micrometers. The method of claim 2,
Said micro space is the said micro container in the cell culture container which has a some micro container on the surface, The area | region in which the said micro container was formed has transparency, The cell culture method characterized by the above-mentioned. The method of claim 5, wherein
The bottom area of the said microcontainer is 9x10 <^-4> mm <^2> -9x10 <^-2> mm <^2> , the height of a side wall is 15 micrometers-300 micrometers, and the side wall width is 3 micrometers-15 micrometers.

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